Herein we report novel protocols for the generation and application of dibromopyridazinediones, an exciting class of disulfide bridging reagents.

This minireview explores published strategies to counteract the retro-Michael instability of the thiosuccinimide linkage, a limiting factor of the efficacy and safety of antibody-drug conjugates.

Diseases are multifactorial, with redundancies and synergies between various pathways. However, most of the antibody-based therapeutics on the market interact with only one target thus limiting their efficacy. The targeting of multiple epitopes could improve the therapeutic index of treatment and counteract mechanisms of resistance. To this effect, a new class of therapeutics emerged: bispecific antibodies.Bispecific formation using chemical methods is rare and low yielding and/or requires a large excess of one of the two proteins to avoid homodimerisation and heterogeneity. In order for chemically prepared bispecifics to deliver their full potential, high-yielding, modular and reliable cross-linking technologies are required. Herein, we describe a novel approach not only for the rapid and high-yielding chemical generation of bispecific antibodies from native antibody fragments, but also for the site-specific dual functionalisation of the resulting bioconjugates. Based on orthogonal clickable functional groups, this strategy enables the assembly of functionalised bispecifics with controlled loading in a modular and convergent manner.

Modification of immunoglobulin G (IgG) 1 proteins in cancer treatment is a rapidly growing field of research. Antibody-drug conjugates (ADCs) exploit the targeted nature of this immunotherapy by conjugating highly potent drugs to antibodies, allowing for effective transport of cargo(s) to cancerous cells. Of the many bioconjugation strategies now available for the formation of highly homogenous ADCs, disulfide modification is considered an effective, low-cost and widely accepted method for modifying IgG1s for improved clinical benefit. However, little is known about how disulfide modification impacts clinically relevant fragment crystallisable (Fc) region interactions. Although often overlooked as a secondary ADC function, Fc interactions could prove key in rational design of cancer cell-targeting ADCs through consideration of potent mechanisms such as antibodydependant cellular cytotoxicity (ADCC). This work explores different IgG1 disulfide modification techniques and the effect they have on quantifiable secondary IgG1 Fc interactions (e.g. CD16a and FcRn). The solvent accessible disulfide residues of trastuzumab, a clinically relevant IgG1, were modified to provide a range of bioconjugates with differing amounts of inter-chain covalent linkages. It was found that by natively re-bridging the IgG1 model, all tested Fc functionalities were not significantly affected. Additionally, in non Fc-specific biophysical experiments (e.g. thermal stability/aggregation), the natively re-bridged species provided an exceptional profile, showing no significant change from the tested native antibody. Conjugates with significant disruption of the covalent connectivity of IgG1 chains resulted in a suboptimal Fc profile (CD16a kinetics or ADCC activity), in addition to sub-standard non Fc-specific attributes (thermal stability). These results advocate native disulfide re-bridging as an excellent synthetic strategy for forming homogenous IgG1 bioconjugates, with no reported negative impact on biophysical profile relative to the native antibody.